结核分支杆菌Mtb8.4真核、原核表达质粒的构建与表达

低分子质量蛋白抗原Mtb8.4是一种非常重要的结核分支杆菌抗原,为了研制结核病核酸疫苗和进行结核病的诊断,分别将其构建到原核和真核载体中进行表达。以结核分支杆菌标准菌株H37Rv基因组DNA为模板,PCR扩增目的基因Mtb8.4,扩增产物酶切后分别克隆到真核表达载体pJW4303和原核表达载体pGEX-4T-1中,构成重组真核表达载体pJW-Mtb8.4和重组原核表达载体pGEX-Mtb8.4,用限制性内切酶消化,PCR扩增及DNA序列分析等多种方法鉴定;并将正确构建的原核表达载体转入E.coliBL21(DE3)plysS中,IPTG诱导表达。结果表明,重组真核表达载体pJW-Mtb8.4和重组原核表达载体pGEX-Mtb8.4构建成功。构建的真核重组质粒pJW-Mtb8.4即可作为结核病DNA疫苗。原核表达载体pGEX-Mtb8.4在BL21(DE3)plysS中成功表达,将表达蛋白进行纯化,作为保护性结核分支杆菌抗原以便检测DNA疫苗的免疫效果。

Construction and Identification of Mycobacterium tuberculosis Mtb8.4 Eukaryotic Expression Plasmid and Prokaryotic Expression Plasmids

The gene encoding the Mtb8.4 protein was amplified based on the genome DNA sequence of H37Rv by PCR and was then cloned into eukaryotic expression vector pJW4303,prokaryotic expression vector pGEX respectively after digesting with restriction endonuclease corresponding to the enzyme sites on the vectors.These recombinant plasmids were identified by means of restricted enzyme digestion, PCR and DNA sequence analysis. Results showed that these plasmids were successfully constructed and the accuracy of these constructs was confirmed. Finally, the prokaryotic recombinant plasmid was transferred into E.coli BL21(DE3)plysS and induced by IPTG to express the protein. This method could be used for the diagnosis of tuberculosis, preparation of subunit vaccine and DNA vaccine and application of those vaccines.